Total RNA was extracted from 5×106 differentiated CD8+ T cells using the NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) isolation kit following the manufacture’s protocol. Total RNA (1 μg) was converted into cDNA using QuantiTect reverse transcription kit (Qiagen, Valencia, CA). Specific primers and probes for qPCR of Gata3, Tbx21, Cyp11a1, Vdr and the housekeeping gene 18SrRNA were designed with Vector NTI advance10 (Life Technologies, Carlsbad, CA) and for Tbx21 a pre-designed assay (Mm00450960_m1, LifeTechnologies, Carlsbad, CA) was used (Supplementary Table 4 ). The determined cycle threshold (Ct) reflects the number of PCR cycles required for the fluorescence signal to exceed the detection threshold, which was set to the log-linear range of the amplification curve. The differences in Ct values of the gene of interest and the house keeping gene 18SrRNA were used to calculate delta Ct (ΔCt). Relative fold changes (RFC) were then calculated using the 2-ΔΔCt algorithm.
Mm00450960 m1
Manufactured by Thermo Fisher Scientific
Sourced in Canada
Mm00450960_m1 is a laboratory product made by Thermo Fisher Scientific. It is a TaqMan Gene Expression Assay used for the detection and quantification of a specific genetic target. The core function of this product is to enable precise gene expression analysis in research applications.
Automatically generated - may contain errors
2 protocols using mm00450960 m1
1
Quantitative RT-PCR Analysis of T-Cell Genes
2
Quantifying T-cell Gene Expression
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Total RNA was extracted from 5 × 106 differentiated CD8+ T cells using the NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) isolation kit following the manufacturer's protocol. Total RNA (1 μg) was converted into complementary DNA using QuantiTect reverse transcription kit (Qiagen, Valencia, CA). Specific primers and probes for qPCR of Gata3, Tbx21, Cyp11a1, Vdr and the housekeeping gene 18SrRNA were designed with Vector NTI advance10 (Life Technologies, Carlsbad, CA) and for Tbx21 a pre-designed assay (Mm00450960_m1, LifeTechnologies) was used (Supplementary Table 4 ). The determined cycle threshold (CT) reflects the number of PCR cycles required for the fluorescence signal to exceed the detection threshold, which was set to the log-linear range of the amplification curve. The differences in CT values of the gene of interest and the house keeping gene 18SrRNA were used to calculate delta CT (ΔCT). Relative fold changes were then calculated using the 2−ΔΔCT algorithm.
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